Shorter J, Lindquist S.
Hsp104 catalyzes formation and elimination of self-replicating Sup35 prion conformers.
Science. 2004 Jun 18;304(5678):1793-7.
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New Study Clarifies How Hsp104 Regulates Yeast Prions and Highlights the Role of Oligomers in Nucleating Amyloid Fibrillogenesis
This paper by James Shorter and Susan Lindquist clarifies the role of the chaperone Hsp104 in both formation and elimination of self-replicating Sup35 yeast prions, called PSI(+). The study also provides insight into the role of soluble oligomers in nucleating amyloid fibril formation. The role of Hsp104 in yeast prion transmission had been enigmatic, because both deletion of Hsp104 or its overexpression eliminates the PSI(+) phenotype. Here, it seems as though either extreme—too little or too much Hsp104—is more effective than a happy medium at eliminating PSI(+).
The N-terminal plus the middle domains (together dubbed NM) of Sup35 are necessary and sufficient for this protein to convert from its soluble state to its prion conformation. The authors show that low concentrations of Hsp104 accelerate NM fibrillization by eliminating the characteristic lag phase of aggregation; specifically by catalyzing the formation of the critical NM oligomers. Soluble oligomers or prefibrillar aggregates precede the appearance of mature fibrils and are a common feature of the amyloid formation pathway for many different types of amyloids.
To directly examine the effect of Hsp104 on NM oligomers, Shorter and Lindquist used an antibody we recently described (Kayed et al., 2003). This conformation-dependent antibody is specific for the oligomeric conformation and does not recognize the monomeric or fibrillar states. Surprisingly, the antibody recognizes an epitope that is common to amyloid oligomers regardless of their sequence. Indeed, Shorter and Lindquist showed that our anti-oligomer antibody also specifically recognizes yeast NM oligomers. In the absence of Hsp104, the species recognized by anti-oligomer antibody peaked late in the lag phase was rapidly consumed during fibril assembly. The authors found that the addition of small amounts of Hsp104 and ATP to soluble NM caused the instantaneous appearance of oligomers that reacted with this antibody, indicating that Hsp104 eliminates the lag phase by catalyzing the formation of NM oligomers that are critical for nucleating fibril assembly. ATP hydrolysis is not required for this effect. Moreover, the oligomer-specific antibody “drastically inhibited unseeded NM polymerization, even when it was 100-fold less abundant,” indicating that the “NM oligomers recognized by the anti-oligomer antibody are crucial for nucleating polymerization.” Since the antibody recognizes a common structure of different amyloid oligomers, this suggests that these oligomers may be generally critical for nucleating other types of amyloid polymerization.
At higher concentrations of Hsp104, NM polymerization was blocked by coupling ATP hydrolysis to the elimination of NM oligomers. When Hsp104 and ATP was added to mature NM fibrils, it caused their disassembly, transiently releasing NM oligomers recognized by anti-oligomer antibody at the early phase of disassembly, suggesting that fibril disassembly initially creates amyloidogenic seeds before it annihilates them. This disassembly activity requires ATP hydrolysis.
These studies have not only clarified the mechanisms by which Hsp104 regulates yeast prions, but additionally have demonstrated that NM oligomers are a critical intermediate for nucleating prion formation. Since these oligomers are common to other amyloids, and are structurally related by virtue of their common interaction with anti-oligomer antibody, their critical role in nucleation of fibrillogenesis may also be a shared property. If this suggestion turns out to be true, the anti-oligomer antibody may provide a simple means of specifically targeting the rate-limiting nucleation step of amyloid fibrillogenesis in a broad range of amyloid-related diseases.
The biphasic effects of the protein-remodeling chaperone Hsp104 on the yeast self-propagating prion [PSI+] phenotype have long been a puzzle. Attempts to explain the mechanism for the elimination of [PSI+] by either deletion or overexpression of Hsp104 have generally invoked a balance of activity and the relative concentrations of conformational states. In this report the authors provide experimental evidence for the differential interaction with, and catalytic action of, Hsp104 on different conformations of NM, the prion domain of Sup35. Low concentrations of Hsp104 generate oligomeric nonfibrillar species that are fibrillogenic, while high concentrations break down these multimeric species and will also disassemble NM fibrils. Disassembly of fibrils, also catalyzed by Hsp104, as well as the disassembly of oligomers requires ATP hydrolysis. Promotion of fibril initiation requires the binding of ATP to Hsp104 but not its hydrolysis. Additionally, while an oligomeric intermediate seeds fibril formation, extension of fibrils is not dependent on species recognized by an oligomer-specific antibody (Kayed et al., 2003). Fibril extension is blocked by a fibril-specific antibody (O'Nuallain and Wetzel, 2002). Hsp70 and its cochaperone Hsp40 do not substitute for Hsp104 on NM in vitro, although they can affect [PSI+] in vivo.
The Hsp100 family of chaperones, which include yeast Hsp104, bacterial ClpB, and plant Hsp110, have been characterized as “molecular crowbars’ (Weibezahn et al., 2004) whose function is to break up large aggregates of proteins formed as a result of environmental stress. Hsp70 and other cellular chaperones are unfolding/refolding machines as opposed to disaggregators. In Hsp100 chaperone-containing cells the disaggregated proteins are cooperatively processed by Hsp70 and other chaperones (Ben-Zvi and Goloubinoff, 2001 ; Glover and Lindquist, 1998). Interestingly, Hsp100 family homologs have not been identified in homeothermic organisms (Neuwald et al., 1999), whose cells may have lost this function over evolutionary time because they do not have to deal with temperature extremes of the external environment. However, they may have traded a higher metabolic rate and constant temperature for susceptibility to accumulation of misfolded proteins. The increased longevity associated with civilization and the concomitant rise in the prevalence of multigenic diseases has been postulated to be caused by “chaperone overload,” pitting accumulating misfolded proteins against protection of silent mutations maintained by chaperone refolding of less stable proteins (Csermely, 2001). The increasing number of late-onset neurodegenerative diseases associated with accumulation of misfolded proteins may reflect this.
The work by Shorter and Lindquist has conceptual implications beyond explaining how Hsp104 can block or stimulate propagation of [PSI+] depending on the relative concentrations of the chaperone and sup35. The experiments with the anti-oligomer and anti-fibrillar conformational antibodies establish that the processes of NM fibril initiation and fibril extension are mechanistically distinct. This divergence has been suspected and is likely for Alzheimer’s Aβ fibril initiation and fibril extension. Initiation of Aβ fibril formation is potently inhibited by Congo Red, while fibril extension is more than 1,000-fold less sensitive. Aβ fibril extension has been shown to occur by monomer addition (Tseng et al., 1999), and ApoE inhibits fibril formation but not extension (Esler et al., 2002).
While inhibition of fibril formation by anti-oligomer antibodies suggests that oligomeric forms of sup35 are obligatory intermediates for fibril formation, the data are insufficient to conclude that the oligomers are on-pathway. It remains to be determined whether the inhibition is kinetic or thermodynamic. Sequestration of an off-pathway oligomeric species kinetically linked to an on-pathway species could give rise to the observed results. Kinetic inhibition by ApoE was observed for Aβ fibril formation (Evans et al., 1995).
The anti-oligomer and anti-fibril antibodies are powerful tools for elucidating conformational states of amyloidogenic proteins of otherwise unrelated primary structure. If the anti-oligomer antibody is effective at 1:100 of the NM concentration, and 10 percent of NM is in an oligomeric state (>100 kDa), either the oligomers are ~10-mers, or only a fraction of the oligomeric NM is capable of nucleating fibril formation. It will be interesting to see if these antibodies or their Fab fragments (to remove the Fc portion that binds Aβ) will allow similar observations to be made for this peptide. The combination of the conformational probes described in this paper with the I41A42-C-terminal substitutions made in Aβ(1-42) (Bitan et al., 2003) should teach us a great deal about the structural basis for Aβ oligomer formation and its relationship to fibril initiation.
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Our unpublished observations suggest that the inhibition of NM fibrillization mediated by the oligomer-specific antibody is in fact thermodynamic and not kinetic. Therefore, we believe that the oligomeric species recognized by the oligomer-specific antibody are most likely to be "on-pathway" for fiber assembly.